Src oncogene at 64B


EVOLUTIONARY HOMOLOGS part 3/3

Src, mitosis and meiosis

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. Components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130(CAS) (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading. Two significant alterations in FAK-mediated signal transduction are found during mitosis: (1) the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16% and 13% of the interphase levels, respectively; (2) mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading (Yamakita, 1999).

A Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), has been purified from oocytes of Xenopus laevis. It has been found that the enzyme is activated within 1 min following fertilization. A number of egg proteins become tyrosine phosphorylated or dephosphorylated within 1 min after fertilization and tyrosine kinase-specific inhibitors can inhibit fertilization-dependent egg activation. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction has also been observed. Parthenogenetic egg activation by a synthetic RGDS peptide, an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. The peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 is inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg is blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner either independent of calcium signaling, or upstream of it (Sato, 1999).

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Data is presented indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. These data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, it was found that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, it is reported that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy (Chu, 2007).

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 310-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases (Grimmler, 2007).

p27Kip1 is an intrinsically unstructured protein (IUP) that controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Usually, binding of p27 inactivates the kinase; however, p27 was surprisingly also found to be associated with active cyclin D holoenzymes. p27 level is frequently controlled by regulated translation and proteolysis. The protein is abundant in quiescent (G0) cells (Hengst and Reed, 1998 and Sherr and Roberts, 1999) and is relatively stable in G0 and early G1 phase. p27 becomes unstable as cells progress toward S phase. p27 degradation is initiated by different ubiquitin ligases. Among these, the KPC1 complex ubiquitinates free unphosphorylated p27, whereas Skp2-dependent E3 ligase complexes target p27 only after phosphorylation on threonine 187 (T187). Active cyclin E/Cdk2 can phosphorylate T187 of cyclin/Cdk-bound p27. While free and active cyclin/Cdk2 efficiently phosphorylates Cdk-bound p27, p27-bound Cdk2 is catalytically inactive due to p27-mediated remodeling of the catalytic cleft and displacement of ATP. This has suggested that degradation by the SCF-Skp2 pathway may require p27-free cyclin E/Cdk2 and has led to the puzzle of how p27 degradation can be initiated in G1 (Grimmler, 2007).

This study reports that p27 can be phosphorylated on a tyrosine residue at position 88 (Y88) within its Cdk-binding domain. This phosphorylation caused the inhibitory 310-helix of p27 to be ejected from the ATP-binding pocket of Cdk2. Y88-phosphorylated p27 still binds to cyclin/Cdk complexes, but the associated kinase retains significant catalytic activity. Furthermore, Y88-phosphorylated p27 becomes an efficient substrate for phosphorylation on T187 by Cdk2 within the trimeric complex. Thus, Y88 phosphorylation may trigger p27 ubiquitination in the absence of free cyclin/Cdk2 and may initiate SCF-Skp2-dependent p27 degradation at the G1/S transition (Grimmler, 2007).

Src dependent modification of cell phenotype and cell fate

Trophoblast giant cell differentiation is characterized by endoreduplication and expression of members of the prolactin (PRL) gene family and can be simulated in vitro via manipulations of the Rcho-1 trophoblast cell line. The regulation of trophoblast cell proliferation and differentiation involves tyrosine protein kinase signaling pathways. Treatment of Rcho-1 trophoblast cells with tyrosine kinase inhibitors disrupts differentiation-dependent expression of members of the PRL gene family and cytoskeletal organization. Activated p60c-src, p62c-yes, and p53/56lyn were present in the Rcho-1 rat trophoblast cell line and in differentiated trophoblast cells isolated from the developing rat placenta. p60c-src and p62c-yes are active in proliferating and differentiating trophoblast cells. During proliferation, p62c-yes exhibits distinct associations with other phosphoproteins (34, 66, 76, and 150 kDa). p53/56lyn is activated only in differentiating trophoblast cells. p53/56lyn shows a differentiation-dependent accumulation in cytoskeletal and membrane fractions, whereas p60c-src levels are virtually invariant in both fractions. Expression patterns of csk (see Drosophila Csk), a negative regulator of Src family kinase activities, are not consistent with its involvement in the differentiation-dependent activation of p53/56lyn; however, there is some indication of the participation of a tyrosine phosphatase in the regulation of p53/56lyn. In conclusion, p60c-src, p62c-yes, and p53/56lyn patterns of activation in trophoblast cells are consistent with their involvement in the control of trophoblast cell proliferation and differentiation (Kamei, 1997).

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), a conditionally immortalized cell line from rat hippocampal neurons (H19-7) was used. Activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocks the induction of differentiation by constitutively activated Raf, the H19-7 cells still undergo differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocks differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocks differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src is sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induces neurite outgrowth. These results suggest that (1) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (2) activation of Src kinases is necessary but not sufficient for differentiation by bFGF, and (3) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src (Kuo, 1997).

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separates this process from NGF-induced neurite outgrowth in PC12 cells (Lazarovici, 1997).

The Src tyrosine kinase has been implicated in a wide variety of signal transduction pathways, yet despite the nearly ubiquitous expression of c-src, src-/- mice show only one major phenotype: osteopetrosis caused by an intrinsic defect in osteoclasts, the cells responsible for resorbing bone. To explore further the role of Src both in osteoclasts and other cell types, transgenic mice have been generated that express the wild-type and mutated versions of the chicken c-src proto-oncogene from the promoter of tartrate resistant acid phosphatase (TRAP), a gene that is expressed highly in osteoclasts. Expression of a wild-type transgene in only a limited number of tissues can fully rescue the src-/- phenotype. Surprisingly, expression of kinase-defective alleles of c-src also reduces osteopetrosis in src-/- animals and partially rescues a defect in cytoskeletal organization observed in src-/- osteoclasts. These results suggest that there are essential kinase-independent functions for Src in vivo. Biochemical examination of osteoclasts from these mice suggest that Src may function in part by recruiting or activating other tyrosine kinases (Schwartzberg, 1997).

Src controls the epidermal growth factor (EGF)-induced dispersion of NBT-II carcinoma epithelial cells. While only Src and Yes are expressed and activated by EGF, microinjected kinase-inactive mutants of Src (SrcK-) and Fyn (FynK-) are able to exert a dominant-negative effect on the scattering response. Both SH2 and SH3 domains of FynK- are required for inhibition of cell scattering. Expression of dominant-negative N17Ras also abrogates EGF-induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK- alters neither the EGF-evoked Shc tyrosine phosphorylation, nor Shc-Grb2 complex formation, nor MAPK activation -- all three are elements of the Ras pathway. Furthermore, the expression of Jun-Fos and Slug rescues the block induced by N17Ras, but not by SrcK-, showing that Src kinases and Ras operate in separate pathways. In addition, actinomycin D inhibition of RNA synthesis represses the ability of the activated mutant L61Ras, but not that of F527Src, to induce epithelial cell scattering. Since tyrosine phosphorylation of cytoskeleton-associated proteins pp125FAK and cortactin are abolished in EGF-stimulated SrcK- cells, it is concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression and by directly regulating the organization of the cortical cytoskeleton (Boyer, 1997).

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. Beta-catenin, gamma-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of alpha-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of alpha-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of beta- and gamma-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn-/- mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of beta-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo (Calautti, 1998).

Src and asymmetric cell division

In early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. These findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell (Bei, 2002).

The mes-1 gene encodes a probable transmembrane protein with overall structural similarity to receptor tyrosine kinase and is a factor required for proper asymmetry and cell fate specification in embryonic germlineage. Null mutations in mes-1 cause a maternal-effect sterile phenotype in which the progeny of homozygous mothers are viable but mature without germcells. Most cell types are specified properly in mes-1 sterile animals, but the germline cell named P4 adopts the fate of its sister cell, a muscle precursor, called D and produces ectopic muscle at the expense of the germline. Interestingly, MES-1 protein is localized intensely at the contact site between the germline blastomere and intestinal precursor at each early developmental stage, starting from the four-cell stage where MES-1 is localized at the contact site between P2 and EMS. An intense phosphotyrosine signal that depends on mes-1(+) activity is correlated with MES-1 protein localization. MES-1 is required in both P2 and EMS and appears to act through a second gene, src-1, a homolog of the vertebrate protooncogene c-Srcpp60. A probable null mutant of src-1 is described that exhibits a fully penetrant maternal-effect embryonic lethal phenotype. The src-1 and mes-1 mutants exhibit similar germline defects and have a nearly identical set of genetic interactions with Wnt/Wg pathway components (Bei, 2002).

Double mutants between mes-1 or src-1 and each of several Wnt/Wg signaling components exhibit a complete loss of P2/EMS signaling, including a loss of the A/P division orientation in the EMS cell. Synergy was observed between mes-1 or src-1 mutants and each of the following previously described mutants: mom-1 (Porcupine), mom-2 (Wnt/Wg), mom-5 (Frizzled), sgg-1 (GSK-3), and mom-3 (uncloned). In addition, identical synergies were observed in the phenotypes of embryos produced by mes-1 or src-1 homozygotes after injection with a mixture of two double-stranded RNAs targeting the C. elegans Disheveled homologs dsh-2 and mig-5. RNAi targeting these Disheveled homologs individually does not induce visible defects in P2/EMS signaling. mes-1 functions in both EMS and P2 to direct MES-1 protein localization at EMS/P2 junction and to specify A/P cleavage orientation in the EMS cell, while src-1 is required cell autonomously in EMS for the induction of the EMS A/P division axis. These findings suggest that a homotypic interaction between MES-1-expressing cells, P2 and EMS, induces a SRC-1-mediated phosphotyrosine signaling pathway that functions in parallel with Wnt/Wg signaling to specify endoderm and to orient the division axis of EMS in early C. elegans embryos (Bei, 2002).

Recent work on dorsal closure in Drosophila has identified a possible convergence between Src and Wnt signaling at the level of regulation of the Jun N-terminal kinase (JNK). Dorsal closure is the process in which epithelial sheets spread over and enclose the dorsal region of the Drosophila embryo during morphogenesis. JNK signaling is essential for dorsal closure and mutants lacking JNK exhibit a dorsal-open phenotype and also exhibit loss of expression of a TGF-ß homolog decapentaplegic in the epithelial cells that lead the closure process. Recent genetic studies have implicated both Src-like kinases and Wnt signaling components in the dorsal closure process and in regulating the expression of dpp. Mutations in Src42A and Wnt signaling factors produce dorsal closure phenotypes similar to JNK mutants and activation of JNK signaling can partially suppress defects caused by these mutants. These findings suggest that Wnt and Src may converge to regulate JNK activity and dorsal closure in Drosophila and thus provide evidence from another system for interactions between these pathways in a developmental process (Bei, 2002).

Multiple Wnt signaling pathways converge to orient the mitotic spindle in early C. elegans embryos: Dshs differentially participate in aligning spindles and vary with respect to their interaction with the Src signaling pathway during spindle orientation

How cells integrate the input of multiple polarizing signals during division is poorly understood. Two distinct C. elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/β-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. CKI (KIN-19) plays a role not only in the Wnt/β-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, a model is established for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event (Walston, 2004).

During development, certain cell divisions must occur with a specific orientation to form complex structures and body plans. In many cases, the polarizing input for oriented divisions involves Wnt signaling. One example of such division involves neuroblasts in Drosophila, in which the first division of the pI sensory organ precursor cell is under the control of Frizzled (Fz) and Dishevelled (Dsh). The orientation of blastomere divisions in the early C. elegans embryo has also been shown to require Wnt signaling. In the 4-cell embryo, the EMS blastomere is induced by its posterior neighbor, the P2 blastomere. This induction has two consequences: it specifies the fates of EMS daughter cells and properly positions the mitotic spindle of EMS. Although both processes are under the control of Wnt signaling, they are controlled through divergent pathways. When EMS divides, the anterior daughter, MS, gives rise to progeny that are primarily mesodermal, and the posterior daughter, E, produces all of the endoderm. The fates of MS and E are controlled in part by a Wnt signaling pathway that regulates the activity of the Tcf/Lef transcription factor, POP-1, in conjunction with the β-catenin WRM-1. WRM-1 interacts with POP-1 through a cofactor, LIT-1, a NEMO-like kinase that is activated through a parallel mitogen-activated protein kinase (MAPK) pathway. Pathways that utilize a β-catenin to alter transcription are referred to as Wnt/β-catenin pathways. Removal of some components of the Wnt/β-catenin pathway alters the fates of the two EMS daughters. Although the fate of the EMS daughters is controlled by a Wnt/β-catenin pathway, the orientation of the EMS division is controlled by a different Wnt pathway (Walston, 2004).

In wild-type embryos, the EMS spindle initially aligns along the left/right (L/R) axis and rotates to adopt an anterior/posterior (A/P) orientation during the initial stages of mitosis. In embryos that lack the function of certain Wnt signaling components, the EMS spindle often sets up in the proper orientation but fails to rotate along the A/P axis until the onset of anaphase. In some cases, the delayed spindle rotates dorsoventrally (D/V) before it adopts the proper A/P alignment. The Wnt spindle orientation pathway that controls EMS orientation involves a Wnt (MOM-2), Porcupine (Porc; MOM-1), and Fz (MOM-5). GSK-3, the C. elegans GSK-3β homolog, has been reported to act positively downstream of the Fz receptor to regulate EMS spindle positioning, rather than as a downregulator of β-catenin accumulation as observed with Wnt/β-catenin signaling. Indeed, Wnt/β-catenin signaling components downstream of GSK-3 are not involved in controlling EMS spindle alignment, and EMS spindle alignment occurs independently of gene transcription. Pathways such as the one that positions the spindle in EMS, which utilize GSK-3 but are independent of transcription, are referred to as Wnt spindle orientation pathways (Walston, 2004).

Although many Wnt signaling components have been identified that participate in spindle orientation, the role of the Dsh family has not been clearly characterized. The Dsh family proteins transmit Wnt signals received from Fz receptors. The Dshs use three domains (DIX, PDZ, and DEP) to interact with different downstream proteins and activate multiple Wnt pathways specifically. The C. elegans genome contains three Dsh family genes that possess the three conserved domains: dsh-1, dsh-2, and mig-5. Transcripts of dsh-2 and mig-5 are at similar, enriched levels in the 4- and 8-cell embryo based on microarray analysis, while dsh-1 levels are low (Walston, 2004).

Another molecule involved in Wnt signaling is Casein Kinase I (CKI). CKI has been shown to prime β-catenin for degradation by phosphorylating it at a specific serine residue. Once primed, the β-catenin can be further phosphorylated and targeted for destruction by GSK-3β. CKI has also been shown to bind and phosphorylate Dsh and may assist in inhibiting GSK-3β when Wnt signaling is active. Loss of function of the CKIα homolog, kin-19, causes defects in the fate of EMS daughter cells. Although the role of CKI in spindle alignment has not been examined, CKIα localizes to centrosomes and mitotic spindles in vertebrate systems (Walston, 2004).

A pathway involving MES-1, a receptor tyrosine kinase, and SRC-1, a Src family tyrosine kinase, acts redundantly with Wnt signaling with respect to the fate of EMS daughters and the orientation of the EMS spindle. When a Src pathway member and a member of the Wnt spindle orientation pathway are removed simultaneously, the EMS spindle fails to rotate into the proper A/P position prior to division and remains misaligned throughout division. Removal of Src pathway members also enhances endoderm fate specification defects observed following removal of Wnt/β-catenin pathway members. Spindle orientation defects in dsh-2(RNAi);mig-5(RNAi) embryos have not been reported unless the Src pathway is also removed; however, only defects in cell division orientation have been reported, as opposed to abnormalities in initial spindle positioning (Walston, 2004).

In addition to regulating the orientation of the EMS division, four of the mom (more mesoderm) genes, mom-1 (Porc), mom-2 (Wnt), mom-5 (Fz), and mom-3 (uncloned), cause spindle alignment defects in the ABar blastomere of the 8-cell embryo. Three of the four AB granddaughters, ABal, ABpl, and ABpr, divide with spindle orientations that are parallel to one another. ABar divides in an orientation that is roughly perpendicular to the other three, an event best viewed from the right side of the embryo, placing anterior to the right. When the function of one of the above mom genes is removed, ABar divides parallel to the other AB granddaughters, resulting in mispositioning of its daughter cells, such that ABarp, the wild-type posterior daughter cell, adopts a position that is anterior to its sister, ABara. The source of the polarizing cue(s) that orients the division of ABar is unclear. However, using blastomere isolations, it has been demonstrated that C, MS, and E are all competent to align the spindle and generate asymmetric expression of POP-1 within unidentified, dividing AB granddaughters, suggesting that one or more of these cells could produce signals that orient the division of ABar in vitro (Walston, 2004).

In this study, the roles of two Wnt signaling pathways involved in regulating the mitotic spindle are demonstrated. (1) The nontranscriptional Wnt spindle alignment pathway requires contact from the C blastomere to align the spindle of ABar. The three Dshs differentially participate in aligning the spindles of EMS and ABar and vary with respect to their interaction with the Src signaling pathway during spindle orientation. Moreover, while KIN-19 participates in endoderm induction through the Wnt/β-catenin pathway, it also acts in the Wnt spindle orientation pathway. (2) A Wnt/β-catenin pathway regulates the timing of spindle rotation in ABar, presumably by specifying the fate of neighboring blastomeres. Taken together, these studies indicate that spindle orientation during early development is a tightly regulated event, influenced by multiple cues transmitted via redundant pathways (Walston, 2004).

Wnt signals in the early embryo are transmitted from P2 to EMS to orient its spindle and to specify the fate of the EMS daughters. The orientation of the spindle relies on Wnt ligands, including MOM-2, that are secreted from P2 and activate MOM-5/Fz on the surface of EMS. This ultimately activates GSK-3, resulting in spindle alignment irrespective of gene transcription or other downstream Wnt/β-catenin components. The current analysis suggests that all three Dsh proteins are upstream of GSK-3 activation. Removal of the function of any of the dshs results in an incorrectly positioned EMS spindle, with varying penetrance. The strongest effect is seen in offspring of dsh-2 mutant mothers, suggesting that DSH-2 is primarily responsible for transducing the signal from MOM-5 to GSK-3 in EMS. Antibody staining shows an enrichment of DSH-2 at the area of cell-cell contact between EMS and P2, consistent with a MOM-2/Wnt signal activating DSH-2 at the cell cortex through the MOM-5/Fz receptor (Walston, 2004).

This analysis also shows that kin-19 contributes to the Wnt spindle orientation pathway in both EMS and ABar. Although KIN-19 participates in EMS fate specification, it has not been demonstrated to influence the orientation of the EMS spindle. Depletion of KIN-19 results in spindle misalignment in EMS and ABar. Additionally, KIN-19 localizes to centrosomes during mitosis: this has been shown to be important in establishing the initial polarization axis in the 1-cell embryo. How kin-19 operates within the pathway remains unclear. Because CKI family members have the ability to prime β-catenin for further phosphorylation by GSK-3, KIN-19 may act as a priming kinase for GSK-3-mediated phosphorylation of other unidentified target proteins. Based on the localization of KIN-19, these targets may be linked to the cytoskeleton, thereby affecting the physical alignment of the spindles of EMS and ABar (Walston, 2004).

This analysis shows that the same Wnt spindle orientation pathway that orients the EMS blastomere also aligns the spindle of the ABar blastomere. The results indicate that, as in EMS, this pathway does not require gene transcription to align the ABar spindle and that GSK-3 could be interacting directly or indirectly with the cytoskeleton (Walston, 2004).

All three dsh genes also act redundantly during ABar spindle orientation as well. Surprisingly, the data show that MIG-5 is the Dsh that is most important during ABar spindle orientation, contrary to the case for EMS spindle alignment, where DSH-2 is most important. The ABar spindle defects seen in dsh-2(or302) embryos suggest that DSH-2 also contributes significantly to ABar spindle orientation. DSH-1 seems to play only a minor role, since dsh-1(RNAi) does not result in ABar spindle defects unless performed along with mig-5(RNAi). This combination may remove enough total Dsh protein to prevent ABar from dividing correctly. In contrast, when dsh-1 function is removed in combination with that of dsh-2, the amount of MIG-5 present may be sufficient to maintain the total Dsh protein at a high enough level that the removal of dsh-1 function has no effect. Alternatively, the Dshs may have slightly different functions in regulating spindle orientation (Walston, 2004).

In Wnt signaling mutants, defective EMS spindle orientation is eventually corrected to the proper orientation, which is presumably due to the activity of the parallel src-1 pathway. In contrast, the Src pathway does not rescue spindle defects in ABar, although the src-1 pathway does influence ABar division. At this time, targets of SRC-1 in spindle orientation are unknown. It is possible that one or more of the Dshs are SRC-1 targets; however, the more severe phenotype of src-1 mutants in EMS suggests that other targets are also affected. Interestingly, in EMS and ABar, removal of src-1 function along with the function of either dsh-1 or mig-5 has very little additional effect on spindle polarity; however, when src-1 function is removed in dsh-2(or302) mutants, spindle misalignment is enhanced to nearly complete penetrance in EMS and ABar. Thus, while the three Dsh proteins act partially redundantly, there may be differences in how they impinge on other pathways (Walston, 2004).

In the 8-cell embryo, ABar contacts the C and MS blastomeres. Blastomere isolations have been used to demonstrate that C and MS can orient the spindle of unidentified AB granddaughters. They also demonstrate that AB granddaughters have random spindle orientation when presented with a mom-2 mutant C blastomere, but not with a mom-2 mutant MS blastomere. Using pal-1(RNAi) to alter the fate of C and laser killing of blastomeres to create steric hindrance within the embryo, ABar has been unambiguously identified. These results show that a loss of contact between C and ABar results in misalignment of its spindle in virtually all cases. Thus, contact with C is not only sufficient to align the spindle of an AB granddaughter but is also necessary to properly orient the ABar spindle through the Wnt spindle alignment pathway. These results further suggest that the polarizing activity of C is mediated by MOM-2/Wnt (Walston, 2004).

The orientation of the EMS spindle is not affected when Wnt/β-catenin signaling is abrogated through disruption of transcription or removal of WRM-1/β-catenin or POP-1/Tcf/Lef. In contrast, when wrm-1, lit-1, pop-1, or ama-1 function is removed, the ABar spindle is delayed in rotating into position. All of these treatments are known to affect the differentiation of the progeny of EMS. Moreover, MS has been shown to be capable of orienting the spindle of AB granddaughters in isolated blastomeres independent of MOM-2 function. Given the physical proximity of the blastomeres to ABar in the wild-type embryo, MS may produce a MOM-2-independent signal that ultimately affects positioning of the ABar centrosome further from C. The data further suggest that abnormalities in the fate of EMS daughters result in rotation defects. In wrm-1(RNAi) embryos, both EMS daughters become MS-like, and β-tubulin::GFP analysis reveals that the centrosomes of ABar do not rotate properly in many cases. If a signal that aids orientation of the spindle of ABar is normally secreted by MS, the two MS-like daughter cells specified in wrm-1(RNAi) embryos could produce competing signals that result in spindle rotation defects in ABar. Similarly, when both of the EMS daughters adopt an E-like fate, as in pop-1(RNAi), altered signaling from EMS daughters could again lead to a similar phenotype. In these cases, the centrosomal positioning presumably relies solely on the Wnt signal from C to eventually position the spindle in the correct orientation (Walston, 2004).

In conclusion, spindle orientation in the early C. elegans embryo is regulated through a Wnt spindle alignment pathway involving the Dshs and KIN-19 but independent of gene transcription. In addition, in ABar, the Wnt/β-catenin pathway regulates the timing of spindle rotation in a transcription-dependent manner, presumably indirectly by altering the fates of E and MS. The components of the Wnt spindle orientation pathway downstream of KIN-19 and GSK-3 are unknown; future work should be aimed at identifying these components and determining which Wnts are involved in specific inductive events (Walston, 2004).

Src and Development

Disabled-1 is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood. Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of the Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. The Dab1 protein level and tyrosine phosphorylation stoichiometry were analyzed by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. Fyn is found to be required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src. It is concluded that Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway (Arnaud, 2003).

Reelin signaling specifies the molecular identity of the pyramidal neuron distal dendritic compartment

The apical dendrites of many neurons contain proximal and distal compartments that receive synaptic inputs from different brain regions. These compartments also contain distinct complements of ion channels that enable the differential processing of their respective synaptic inputs, making them functionally distinct. At present, the molecular mechanisms that specify dendritic compartments are not well understood. This study reports that the extracellular matrix protein Reelin, acting through its downstream, intracellular Dab1 (see Drosophila Dab) and Src family tyrosine kinase signaling cascade, is essential for establishing and maintaining the molecular identity of the distal dendritic compartment of cortical pyramidal neurons. Reelin signaling is required for the striking enrichment of HCN1 and GIRK1 channels in the distal tuft dendrites of both hippocampal CA1 and neocortical layer 5 pyramidal neurons, where the channels actively filter inputs targeted to these dendritic domains (Siegelbaum, 2014).

PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A. However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαq subunits and protein kinase C (PKC). In CA1 neurons, PACAP38 enhances synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons (Macdonald, 2005).

The role of Src in long-term potentiation

Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that is considered to be a model of learning and memory. Protein tyrosine phosphorylation is necessary to induce LTP. Induction of LTP in the CA1 pyramidal cells of rats is prevented by blocking the tyrosine kinase Src, and Src activity is increased by stimulation that produces LTP. Directly activating Src in the postsynaptic neuron enhances excitatory synaptic responses, occluding LTP. Src-induced enhancement of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor-mediated synaptic responses requires raised levels of intracellular Ca2+ and N-methyl-D-aspartate (NMDA) receptors. Thus, Src activation is necessary and sufficient for inducing LTP and may function by up-regulating NMDA receptors (Lu, 1998).

Src and transformation

Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. An experimental model of oncogenesis is described involving a derivative of MCF10A, a spontaneously immortalized cell line derived from normal mammary epithelial cells, that contains ER-Src, a fusion of the Src kinase oncoprotein (v-Src) and the ligand binding domain of the estrogen receptor. Treatment of these cells with estrogen receptor antagonist tamoxifen (TAM) for 36 hr results in phenotypic transformation, formation of multiple foci, the ability to form colonies in soft agar, increased motility and invasive ability, and tumor formation upon injection in nude mice. This model permits the opportunity to kinetically follow the pathway of cellular transformation in a manner similar to that used to study viral infection and other temporally ordered processes. Transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal (Iliopoulos, 2009).

c-Src drives intestinal regeneration and transformation

The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. This study used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, it was demonstrated that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, it was shown that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, this study demonstrated a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo (Cordero, 2014).

A noncanonical Frizzled2 pathway regulates epithelial-mesenchymal transition and metastasis

Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. This study found that the Wnt receptor Frizzled2 (Fzd2; see Drosophila Frizzled) and its ligands Wnt5a/b (see Drosophila Wingless) are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3. A gene signature regulated by this pathway predicts metastasis and overall survival in patients. An antibody was developed to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. It is proposed that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b (Gujral, 2014).

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Src oncogene at 64B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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